Each of the references discussed in this specification are expressly incorporated by reference in their entirety, whether or not specifically mentioned in association therewith.
“Click chemistry” is a technology first developed by Sharpless at Scripps Institute, employing a copper (I) catalyzed reaction in which azide moieties are attached via a 1,3-cycloaddition with an alkyne. Copper-free click chemistry reactions are also possible. Methods of using click chemistry are known in the art and include those described by Rostovtsev et al., Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., Bioconjugate Chem., 2006, 17, 52-57, Baskin et al., Proc. Natl. Acad. Sci. USA 2007, 104, 16793-16797). Unfortunately, the mandatory copper catalyst is toxic to both bacterial and mammalian cells, thus precluding applications wherein the cells must remain viable. Catalyst-free Huisgen cycloadditions of alkynes activated by electron-withdrawing substituents have been reported to occur at ambient temperatures. However, these compounds undergo Michael reaction with biological nucleophiles.
“Click Chemistry” reactions are high yielding, wide in scope, create only byproducts that can be removed without chromatography, simple to perform, and can be conducted in easily removable or benign solvents. This concept was developed in parallel with the interest within the pharmaceutical, materials, and other industries in capabilities for generating large libraries of compounds for screening in discovery research. Several types of reaction have been identified that fulfill these criteria, thermodynamically-favored reactions that lead specifically to one product, such as nucleophilic ring opening reactions of epoxides and aziridines, non-aldol type carbonyl reactions, such as formation of hydrazones and heterocycles, additions to carbon-carbon multiple bonds, such as oxidative formation of epoxides and Michael Additions, and cycloaddition reactions.
The azide-alkyne cycloaddition fulfills many of the prerequisites. Many of the starting monosubstituted alkynes and organic azides are available commercially, many others can easily be synthesized with a wide range of functional groups, and their cycloaddition reaction selectively gives 1,2,3-triazoles. See FIG. 45.

Unfortunately, the thermal Huisgen 1,3-Dipolar Cycloaddition of alkynes to azides requires elevated temperatures and often produces mixtures of the two regioisomers when using asymmetric alkynes. In this respect, the classic 1,3-dipolar cycloaddition fails as a true “click” reaction. A copper-catalyzed variant that follows a different mechanism can be conducted under aqueous conditions, even at room temperature. Additionally, whereas the classic Huisgen 1,3-dipolar cycloaddition often gives mixtures of regioisomers, the copper-catalyzed reaction allows the synthesis of the 1,4-disubstituted regioisomers specifically. By contrast, a later developed ruthenium-catalyzed reaction gives the opposite regioselectivity with the formation of 1,5-disubstituted triazoles. Thus, these catalyzed reactions comply with the definition of click chemistry and have put a focus on azide-alkyne cycloaddition as a prototype click reaction. See FIG. 46.

The click chemistry reactions are reactions between two functional groups that are highly selective, easy to perform and proceed at nearly quantitative yield. A biocompatible click reaction must have these characteristics but in addition such reactions need to proceed in dilute aqueous solution at neutral pH, and preferably with rapid kinetics. A bioorthogonal click reaction possesses all the characteristics of a biocompatible reaction with the additional requirement that the reactive functional groups do not form stable covalent bonds with functionalities in the biological system.
One of the first types of reaction that was used for bioorthogonal ligations is the reaction of aldehydes and ketones with heteroatom-bonded amines that are sometimes called “α-effect amines” (hydrazine, hydrazide, and other amine-nitrogen compounds, aminooxy reagents, etc.). While an amine-aldehyde/ketone reaction is highly reversible in aqueous solution, an α-effect amine-aldehyde/ketone reaction proceeds readily in mildly acidic aqueous solution to form a product (hydrazone or oxime) that is considerably more stable and less reversible than the product formed with a simple amine. The characteristics of the reaction are sharply dependent on the precise nature of α-effect amine and the carbonyl-containing reactive partner. Such reactions have been well studied. (Jencks, W. P. (1969) Catalysis in Chemistry and Enzymology (McGraw-Hill Series in Advanced Chemistry), McGraw-Hill.)
The alpha effect refers to the increased nucleophilicity of a molecule due to the presence of an adjacent (alpha) atom with lone pair electrons. These include hydrazine, hydroxylamine, the hypochlorite ion and the hydroperoxide anion. (Buncel, E., and Um, I.-H. (2004) The α-effect and its modulation by solvent, Tetrahedron 60, 7801-7825.)
A major drawback to the use of aldehyde-ketone condensation reactions with α-effect amines is that the optimal pH of the reaction is normally 2-5, which is much lower than physiological pH. (Smith, P. A. S. (1982) Derivatives of Hydrazine and Other Hydronitrogens Having N—N Bonds, The Benjamin/Cummings Publishing Company, London.) Attempts to speed up the reactions at neutral pH include addition of an aromatic amine as a catalyst. Other efforts have focused on changing the structure of the nucleophile (hydrazine) or the substituents on an aromatic aldehyde, including adding an intramolecular proton source for catalysis. (Dirksen, A., Hackeng, T. M., and Dawson, P. E. (2006) Nucleophilic catalysis of oxime ligation, Angewandte Chemie-International Edition 45, 7581-7584. Kool, E. T., Crisalli, P., and Chan, K. M. (2014) Fast Alpha Nucleophiles: Structures that Undergo Rapid Hydrazone/Oxime Formation at Neutral pH, Org. Lett. 16, 1454-1457.)
Another drawback of aldehyde-ketone condensation reactions with α-effect amines is the product may not be sufficiently stable under physiological conditions. (Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D., and Bertozzi, C. R. (2013) A Pictet-Spengler ligation for protein chemical modification, Proc. Natl. Acad. Sci. USA 110, 46-51). However, the vast majority of examples use the hydrazone and oxime-forming reactions mentioned previously because of their bioorthogonality, operational simplicity (i.e., no auxiliary reagents are required), and good yields under mild aqueous conditions. However, the resulting C═N bonds are susceptible to hydrolysis (Mueller B M, Wrasidlo W A, Reisfeld R A. Antibody conjugates with morpholinodoxorubicin and acid-cleavable linkers. Bioconjug Chem. 1990; 1(5):325-330.), undermining the use of such conjugates in situations in which long-term stability is required. The oxime has been identified as the most hydrolytically stable C═N linkage, but it is still thermodynamically unstable to hydrolysis under dilute conditions, decomposing via an acid-catalyzed process (Kalia J, Raines R T. Hydrolytic stability of hydrazones and oximes. Angew Chem Int Ed Engl. 2008; 47(39):7523-7526). Many researchers have found that oxime conjugates that are kept under ideal storage conditions—low temperature, high concentration, and neutral or high pH—are kinetically stable and are therefore suitable for short-term laboratory studies (Hudak J E, Yu H H, Bertozzi C R. Protein glycoengineering enabled by the versatile synthesis of aminooxy glycans and the genetically encoded aldehyde tag. J Am Chem Soc. 2011; 133(40):16127-16135; Shi X, et al. Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging. Nat Methods. 2012; 9(5):499-503; Yi L, et al. A highly efficient strategy for modification of proteins at the C terminus. Angew Chem Int Ed Engl. 2010; 49(49):9417-9421.). However, biological applications requiring extended persistence of the conjugate at physiological temperatures and low concentrations necessitate a significantly more stable covalent linkage than the oxime provides.
Bioorthogonal reactions are chemical reactions that can be used in biological systems, coupling one reactive group specifically with another reactive group: without side reactions; in neutral, aqueous solution; and under additional conditions that are compatible with the biological system. Bioorthogonal reactions can be used for conjugating a biomolecule and a reporter; in biotechnology; proteomics; (bio)polymer engineering; sensors and detectors; and drug delivery. See C&E News, www.cen-online.org/articles/89/i34/Unnaturally-Productive.html; Lahann, J., (Ed.) (2009) Click Chemistry for Biotechnology and Materials Science, John Wiley & Sons Ltd, West Sussex, UK., Viral Nanoparticles: Tools for Materials Science & Biomedicine, Marianne Manchester, Nicole Franziska Steinmetz; Macromolecules 2010, 43, 1.
The ideal bioconjugation chemistry has, for example, the following characteristics, some of which are optional or context dependent (adapted from Solulink, Inc. White paper on Bioconjugation Chemistry):
a) linkers must be incorporated on biomolecules in a mild, controllable manner
b) the inherent biological function of the biomolecules must be unaffected after modification and conjugation
c) the conjugation reaction occurs directly upon mixing the two modified biomolecules, preferably not requiring addition of an oxidant, reductant, or metal.
d) modified biomolecules are stable over extended periods
e) conjugation occurs in buffered aqueous solutions, at a physiological pH
f) stoichiometrically efficient (e.g., 1:1)
g) fast reaction kinetics
h) no undesirable covalent side reactions during modification
i) linkers can be incorporated on a variety of biomolecules, including oligonucleotides and peptides.
The concept of bioorthogonality means that the technology does not interfere with biological processes in the same medium (unless specifically targeted), and the technology is not interfered with by components of the biological medium. Bioorthogonal processes therefore occur in aqueous medium, without addition of toxic substances (or toxic concentrations of substances), within a physiological pH range (e.g., ˜6-8), at physiological temperatures (e.g., 0-42° C., depending on species) and pressures (e.g., 1 Atm), are not interactive with physiological thiols or amines, or are sensitive to redox chemistry. Further, the biological environment typically contains a range of enzymes that can degrade certain structures, and therefore bioorthogonal reaction reagents or products should not be sensitive to modification by the various enzymes in the medium. See, Bertozzi: Chem Soc Review, 2010; US 2011/0207147.
Click chemistry reactions have applications beyond biological systems, including materials chemistry. (Iha, R. K., Wooley, K. L., Nystrom, A. M., Burke, D. J., Kade, M. J., and Hawker, C. J. (2009) Applications of Orthogonal “Click” Chemistries in the Synthesis of Functional Soft Materials, Chemical Reviews 109, 5620-5686; Oommen, O. P., Wang, S., Kisiel, M., Sloff, M., Hilborn, J., and Varghese, O. P. (2013) Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering, Advanced Functional Materials 23, 1273-1280.).
See also,
Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality Ellen M. Sletten and Carolyn R. Bertozzi Angew. Chem. Int. Ed. 2009, 48, 6974-6998;
Bioorthogonal chemistry: recent progress and future directions Reyna K. V. Lim and Qing Lin Chem. Commun., 2010, 46, 1589-1600;
Dehydration Reactions in Water. Brønsted Acid-Surfactant-Combined Catalyst for Ester, Ether, Thioether, and Dithioacetal Formation in Water JACS 124 11971 2002 (“Although various efficient catalytic systems in water have been developed so far, there are still many types of reactions which are difficult to carry out in water. One such reaction is dehydration in which water molecules generated during the reaction must be removed to shift equilibrium to the dehydrated product side. A representative example is acid-catalyzed direct esterification of carboxylic acids with alcohols. Generally, direct esterification is carried out in organic solvents and requires either of two methods to shift the equilibrium to afford the product (ester) in good yields: continuous removal of water during the reaction (azeotropically or using dehydrating agents) and use of a large excess of one of the reactants. In any case, the presence of large excess amounts of water as a solvent should have a detrimental effect on the equilibrium of the dehydration reaction.”);
Organic chemistry in water Chem. Soc. Rev., 2006, 35, 68-82 (“Dehydration is a very common reaction in organic chemistry. It is difficult to carry out in water because water molecules generated during the reaction must be removed to shift equilibrium toward the side of the dehydrated product.”);
Organic chemistry in water Chem. Soc. Rev., 2006, 35, 68-82 (“Aqueous organic chemistry is essential for the emerging field of chemical biology, which uses chemical tools to study biology. Since life constructs chemical bonds in aqueous environments, selective chemical reactions designed to modify biomolecules are now recognized as powerful tools in chemical biology. They provide insight into cellular processes and inspire new strategies for protein engineering. To achieve this goal, the participating functional groups must have a narrow distribution of reactivity and must be inert toward biological molecules. In addition, the selective chemical reactions must occur at room temperature and in aqueous physiological environments.”);
Planar Boron Heterocycles with Nucleic Acid-Like Hydrogen-Bonding Motifs Michael P. Groziak, Liya Chen, Lin Yi, and Paul D. Robinson, J. Am. Chem. Soc. 1997, 119, 7817-7826;
Biological and Medicinal Applications of Boronic Acids Wenqian Yang, Xingming Gao, and Binghe WangBoronic Acids. Edited by Dennis G. Hall Copyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30991-8;
Aminoboronic acids and esters: from synthetic challenges to the discovery of unique classes of enzyme inhibitors Chem. Soc. Rev., 2011, 40, 3895-3914;
Monoclonal antibody-based therapies in cancer: Advances and challenges. Pharmacology & Therapeutics 138 (2013) 452-469 (“Over the last decade of ADC (antibody-Drug Conjugates) development, it has become clear that choice of conjugation strategy and sites on the Ab are highly important in determining the tolerability, pharmacokinetic (PK) properties and overall effectiveness of ADC therapy. Ideally, conjugation of the Ab to the drug should not perturb the integrity of the Ab, the binding of the Ab to the antigen, or the biological activity of the drug.”) A method for putting hydrazides on antibodies (Immunoliposomes: Targeted Delivery—Hydrazide Modification) is described. See FIGS. 37 and 38;
Labeling Strategies of Peptides with 18F for Positron Emission Tomography. Current Topics in Medicinal Chemistry, 2010, 10, 1669-1679 (“The field of 18F-fluorine chemistry applied on peptides is expanding. A variety of recently employed labeling strategies like acylation, alkylation, thiol reactive, oxime formers, 1,3-dipolar cycloadditions have been summarized in this review. Higher yields, milder reaction conditions and simplification for automation are important drivers for the ongoing development. The simultaneous elevation in understanding of 18F labeling mechanisms makes hopes for the clinical usefulness of radiolabeled peptides for human diagnostic medicine and therapy monitoring.”);
Click-Chemistry Reactions in Radiopharmaceutical Chemistry: Fast & Easy Introduction of Radiolabels into Biomolecules for In Vivo Imaging Current Medicinal Chemistry, 2010, 17, 1092-1116 (“When introducing radioactive nuclides with a very short half-life into biomolecules . . . Time is always the most important issue . . . This is the reason why just a part of the reactions that belong in principle to the group of click reactions have shown to meet the requirements for radiosyntheses.”)(Lahann, J., (Ed.) (2009) Click Chemistry for Biotechnology and Materials Science, John Wiley & Sons Ltd, West Sussex, UK.);
Bioorthogonal Chemistry for Site-Specific Labeling and Surface Immobilization of Proteins Yong-Xiang Chen, Gemma Triola, and Herbert Waldmann, Acc. Chem. Res., 2011, 44 (9), pp 762-773;
Chemically modified viruses: principles and applications Kristopher J Koudelka and Marianne Manchester Current Opinion in Chemical Biology 2010, 14:810-817 (“Viral nanotechnology is a highly interdisciplinary field, incorporating virology, chemistry, physics, physiology, pharmacology, and materials science [1]. Methods have been established for the efficient production and chemical modification of viral nanoparticles (VNPs), as well as non-infectious virus-like particles (VLPs) that mimic the structure of infectious particles but lack nucleic acid. These methods provide the foundation for fine-tuning of ligand or probe attachment, immobilization of VNPs on surfaces, or assembly into complex aggregate or network structures . . . . The ability to precisely place components on the surface of viruses, by chemical or genetic means, has allowed for the creation of complex systems that impart novel function.”);
Click chemistry by microcontact printing on self-assembled monolayers: A structure-reactivity study by fluorescence microscopy Jan Mehlich and Bart Jan Ravoo. Org. Biomol. Chem., 2011, 9, 4108 (“The modification of inorganic surfaces with monolayers of organic molecules has found widespread application in nanofabrication, sensing, diagnostics and molecular electronics.1-6 The microscale patterning of molecular monolayers is crucial to all of these applications. In recent years, microcontact printing (mCP) has developed into a powerful tool to functionalize substrates with spatially patterned molecular monolayers . . . . Recently, it was shown that also the Huisgen 1,3-dipolar cycloaddition of alkynes and azides can be induced by mCP.17 It was demonstrated that the cycloaddition by mCP proceeds to completion (i.e. until all reactive sites on the surface are occupied) within a few hours when a Cu-coated stamp is used or Cu(I) catalyst is added to the alkyne ink.”);
Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives. Journal of Controlled Release 53 (1998) 93-103 (“Hyaluronic acid (HA), The immunoneutrality of HA makes it an HA [6-9]. HA ranging in size from six disaccharide excellent building block for biomaterials to be employed for tissue engineering and drug delivery. Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.”);
Dendritic sugar-microarrays by click chemistry Tomohiro Fukuda, Shunsuke Onogi, Yoshiko Miura Thin Solid Films 518 (2009) 880-888;
Applications of Orthogonal “Click” Chemistries in the Synthesis of Functional Soft Materials Chem. Rev. 2009, 109, 5620-5686;
Lahann, J., (Ed.) (2009) Click Chemistry for Biotechnology and Materials Science, John Wiley & Sons Ltd, West Sussex, UK. (From Preface: “While the concept of click chemistry might have initially been introduced with a firm eye on drug discovery, its applications to materials synthesis and biotechnology have been a startling success story. Thus, as I look ahead toward the advances coming from click chemistry in the next decade, some of the most promising applications are related to materials science and biotechnology. With this book, it is my intention to share some of the excitement surrounding click chemistry by describing the most recent progress with respect to (i) the development of a conceptual framework of click chemistry, (ii) its application to the precise design and synthesis of macromolecules, and (iii) its numerous applications in materials science and biotechnology.”);
Jencks, W. P. (1969) Catalysis in Chemistry and Enzymology (McGraw-Hill Series in Advanced Chemistry), McGraw-Hill;
Buncel, E., and Um, I.-H. (2004) The α-effect and its modulation by solvent, Tetrahedron 60, 7801-7825;
Smith, P. A. S. (1982) Derivatives of Hydrazine and Other Hydronitrogens Having N—N Bonds, The Benjamin/Cummings Publishing Company, London;
Dirksen, A., Hackeng, T. M., and Dawson, P. E. (2006) Nucleophilic catalysis of oxime ligation, Angewandte Chemie-International Edition 45, 7581-7584;
Kool, E. T., Crisalli, P., and Chan, K. M. (2014) Fast Alpha Nucleophiles: Structures that Undergo Rapid Hydrazone/Oxime Formation at Neutral pH, Org. Lett. 16, 1454-1457;
Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D., and Bertozzi, C. R. (2013) A Pictet-Spengler ligation for protein chemical modification, Proc. Natl. Acad. Sci. USA 110, 46-51;
Lahann, J., (Ed.) (2009) Click Chemistry for Biotechnology and Materials Science, John Wiley & Sons Ltd, West Sussex, UK;
Iha, R. K., Wooley, K. L., Nystrom, A. M., Burke, D. J., Kade, M. J., and Hawker, C. J. (2009) Applications of Orthogonal “Click” Chemistries in the Synthesis of Functional Soft Materials, Chemical Reviews 109, 5620-5686;
Oommen, O. P., Wang, S., Kisiel, M., Sloff, M., Hilborn, J., and Varghese, O. P. (2013) Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering, Advanced Functional Materials 23, 1273-1280;
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